Introduction

Contrary to how it may be taught in some laboratory classrooms, the process of scientific inquiry encompasses much more than the collection and interpretation of data. A key part of the process is design – of experiments that specifically address a hypothesis and of new materials or technologies. Moreover, any design is subject to continued revision. You might redesign an experiment or tool based on your own own research, or you might consult the vast body of scientific literature for other perspectives. As the old graduate student saying goes, “A month in the lab might save you a day in the library!” In other words, although the process of combing the literature can be arduous or even tedious at times, it beats wasting a month of your time repeating experiments already proven not to work or reinventing the wheel.

During this module, each of you will design and test a modification to a biological system. The parent system is a light-based edge detector genetically encoded in bacteria. You may be wondering why on earth such a thing would exist! The edge detector is not a product of nature — not entirely at least — but one of synthetic biology. Scientists in this relatively young field aspire to reliably program cells to carry out arbitrary functions. (Where by arbitrary I mean "unconstrained" rather than "capricious," though sometimes both apply.)

The edge detection system comprises several sub-systems that we can call modules. Taking a modular approach is useful in programming biology, just as it is in programming computers. To understand how the complete system works, it may be helpful to examine these modules one at a time, as well as to discuss a similar system that detects and copies an entire image rather than only its edges. In both systems, the final output is a black precipitate created when beta galactosidase (β-gal) cleaves 3,4-cyclohexenoesculetin-b-D-galacto- pyranoside (S-gal). A number of genetic elements control when and whether β-gal is produced.

The original bacterial photography system was described in 2005 by Levskaya et al. in the Voigt lab. In this system the gene lacZ, which encodes β-gal, is under the control of the ompC promoter. We will discuss how this promoter works in detail later on; for now, you need only understand that in the presence of red light the promoter is off, and in darkness it is on (producing β-gal). The genetic circuit depiction of the system is below. Note that besides a light-sensitive ompC promoter, the bacteria also carry plasmids for making an accessory light-sensing protein called phycocyanobilin (PCB).

Genetic circuit for bacterial photography system, from Levskaya et al. Note that the promoter is on when phosphorylated, which happens in the dark. You don't need to understand all the details of how this promoter works today.

These genetically engineered bacteria can be grown in a special medium containing a source of ferric ion along with S-gal, a synthetic analogue of the natural substrate for β-gal that produces a black precipitate. Thus, when a black-and-transparent mask is placed over a Petri dish containing these genetically engineered bacteria, a black precipitate is formed by the bacteria under the dark part of the mask, but not by the bacteria under the transparent part. An example of such a mask and its corresponding bacterial photograph is shown below.

In the edge detection system, described by Tabor et al. in 2009, lacZ is not directly under the control of PompC, but is separated from it by an intermediate module. Now when PompC is on, it drives expression of both luxI and cI genes. Meanwhile, luxR is constitutively expressed. Again, we will discuss the details of the biology later on, but for now you should understand the following: luxI makes AHL, a diffusible signal that together with LuxR protein turns on the hybrid lux-lambda promoter (Plux-λ); cI makes a (non-secreted) protein called lambda repressor that dominantly represses Plux-λ; finally, Plux-λ controls lacZ expression. The diagram below depicts the genetic circuit. Now, what does all this mean for bacteria growing below a photomask?

Genetic circuit for edge detection system, from Tabor et al.

Bacteria that are in the dark produce both AHL and lambda repressor, but due to the latter’s dominant action do not produce β-gal. Bacteria that are in the light produce neither AHL nor repressor, and thus are free to respond to the AHL produced by cells in the dark. So long as the AHL only has time to diffuse to the light-dark border, a crisp black line is produced there, hence detecting and copying the edge of the image. In reality, some background β-gal production may occur in other regions of the plate, though it is strongest at the light-dark border. Below is an edge detection image made using the mooing turtle mask. Note the reversal of light and dark areas, and the somewhat pronounced darker edges.

Bacterial edge detector image of mooing turtle.

Today you will spend much of your time getting intimately familiar with the edge detection system, along with a derivative system that is responsive to a small molecule rather than to light. To get a physical sense of what will otherwise be a very abstract couple of days, you will gather some data about these two systems. You will prepare an edge detection plate today and see the poor contrast of the original (light-based) system for yourself next time. Note that plating cells on solid media is materials-intensive and that β-gal production in the plate is not easily quantifiable. Growing cells in liquid culture allows for easier and more reproducible quantification. However, cells that exist just on the light side of the light-dark border in the plate must be approximated in liquid culture by exogeneously adding AHL. In your case, you will prepare liquid cultures for the derivative (IPTG-based) system under four conditions, and compare the results you get next time to the truth table you are given today.

Protocols

Part 1: Explore system components

Promoter background/refresher

To prepare for designing a modification to the edge detection system, you should first refresh your memory (or take a crash course in) genetic control elements. You want to understand the functions of promoters, operators, and ribosome binding sites. One resource for learning about genetic control elements in the context of synthetic biology is the Registry of Standard Biological Parts. The registry was developed as one strategy to make routinely engineering biology feasible: just as mechanical and electrical engineers have access to defined parts that perform in predictable ways together, so should biological engineers.

You can learn the definition and basic structure of promoters and operators here, and learn more about promoter regulation here. Ribosome binding sites are described here and here. More detail about consensus promoter sequences is contained in the attached lecture notes from a class at Memorial University in Canada.

Get familiar with original edge detection system

Start by drawing the edge detector circuit in your notebook. Include genes, promoters, and relevant gene products. Although this may seem like a silly or redundant exercise, perhaps nothing makes circuit abstractions understandable better than drawing them out with your own two hands. (Or even just the one hand!)

One way to think of genetic circuits is as systems with binary inputs and outputs. As we will find later, simple on and off states may not fully describe our system; partially on states may exist. For now, you should fill in the table below with 0's (for absent/off) and 1's (for present/on) for the four possible conditions in which our two-input system can exist. Such a description is called a truth table.

For each entry in the table, write a sentence or two in your notebook explaining your reasoning.

Now consider a population-level view of the system. Draw a box with a line through it, and label one half "light" and the other "dark." The box represents a Petri dish full of cells (but with an easier geometry!). In the lab, the light and dark regions can be made by using a mask with transparent and opaque regions.

Where is AHL being produced?

Over short times, how far from where it is produced might the AHL diffuse? Shade that region.

For each entry in the truth table, which region of the box is represented (light, dark, or shaded)?

INPUTS

OUTPUTS

Light

AHL

AHL

lacZ

What-if-scenarios

The truth table above is a high-level view of our system. It does not take into account the effect of leaky promoters, RBS of more-than- or in-sufficient strength, or mismatched elements. Consider how the following scenarios would affect your truth table. Write partially on states as (p) for partial, and again briefly explain your reasoning in your notebook.

What if POmpC is slightly leaky, and cI is preceded by a very strong RBS?

What if Plux-λ has very high basal expression?

What if leaky expression from POmpC produces relative amounts of AHL and cI such that cI cannot fully repress?

What if cI is mutated such that it is very difficult for the repressor protein to form active dimers?

The real system

In fact, the truth table for the edge detector system looks like the following. Explain what two problems the system appears to have. (Hint: two different elements are leaky.) Note that we only measured production of β-galactosidase, so AHLout is marked "nm" for not measured.

INPUTS

OUTPUTS

Light

AHL

AHL

lacZ

0

0

nm

(p), weak

0

1

nm

(p), weak

1

0

nm

(p), strong

1

1

nm

1

Get familiar with model (IPTG) system

We designed an auxiliary testing system to the edge detector that is sensitive to a small molecule - IPTG - rather than light. This system provides two major advantages. First, it is more tenable to work with a small molecule than with light, especially for multiple samples. Even the Voigt lab had to spend 5 days getting some crucial data for their paper (1 day per light intensity!), whereas we can readily test different IPTG concentrations in parallel. Second, and more crucially, this design allows us to isolate one system imperfection at a time, as you will see below.

The genetic circuit depiction of the IPTG-based system is shown below. Recall that lacI encodes the lac repressor protein, which turns off plac. In the presence of lactose or a small-molecule lactose analogue such as IPTG (isopropyl β-D-thiogalactoside), the repressor is prevented from binding to its associated operator, and the lac promoter is on. Note that placIq is a constitutive promoter.

IPTG-sensitive model system, modified from Tabor, et al.

Again begin by drawing the circuit in your lab notebook, and make sure you understand how the elements interact.

Map the IPTG- to the light-sensitive system. Is IPTG equivalent to light or NOT light?

The truth table for our pseudo-edge detector looks like the following. Can you rationalize why?

INPUTS

OUTPUTS

IPTG

AHL

AHL

lacZ

0

0

nm

(p), medium-high

0

1

nm

1

1

0

nm

0 or very weak (p)

1

1

nm

0 or very weak (p)

Note that our model system is just a tool for fixing the original system. This limited utility is because the IPTG-sensitive system has no inherent spatial element; we cannot simply put a mask on the culture plate to create areas of no IPTG and +IPTG, the way we can for light and dark. (At least not as trivially! Of course we could imagine designing such a system.)

For homework you will begin to explore in detail one of the leaky elements that you identified above. Next time in lab you will design a modification to it in order to reduce leakiness.

Part 2: Prepare model system liquid cultures

If you are waiting for the spectrophotometer, you can begin by picking up eight 14 mL round-bottom tubes and preparing sticky labels for your samples (described in step 6).

Measure the OD600 value of a 1:10 dilution of your cells (use a total volume of 600 μL).

OD stands for optical density and is a measure of light scattered by the cells in your culture. It increases with increasing cell density.

Everyone should measure about the same OD - compare with another lab group to check your work.

In a 50 mL conical tube, add 25 μL of ampicillin to 25 mL of LB medium and invert briefly to mix.

Dilute the cell stock to an OD of 0.0025 in the 25 mL volume.

Remember to take into account that you measured a diluted sample!

You can treat the cell volume as negligible.

Distribute 2.5 mL of cells to each round-bottom tube with a 5 or 10 mL serological pipet.

Distribute additives to each tube according to the scheme below, making sure that each tube is well-labeled:

Two tubes get nothing further.

Two tubes get 2.5 μL of AHL (1000X stock).

Two tubes get 12.5 μL of IPTG (1 M stock). (What is the final IPTG concentration?)

Two tubes get both AHL and IPTG.

Make sure each cap is snapped shut. You will hear a "snap" when it's really closed. If you're uncertain whether your tubes are closed, please ask one of the teaching faculty.

Ask an instructor to show you how to operate the roller wheel in the incubator.

Your cultures will be grown overnight, then moved to 4 °C until next time.

Part 3: Prepare edge detection plate

Some media has been prewarming in the 42 °C waterbath for you. The media is traditional LB supplemented with ferric ammonium citrate and S-gal. When β-gal cleaves S-gal in the presence of iron, a black precipitate forms. The media also has agarose so it will harden as it cools; the low melting point of the agarose allows us to suspend cells in the media without killing them from excessive heat. All manipulations should be with your best sterile technique to avoid contamination of your cultures.

Before you begin you should pick up an empty Petri dish, empty 15 mL conical tube tube, and a transparency with a printed design from the front bench.

Add 45 μl of antibiotic cocktail (equal volumes mix of 1000X ampicillin, chloramphenicol, and kanamycin) to the tube. The antibiotics will maintain the three edge detection plasmids in the bacteria as the cells grow.

Pour 15 ml of molten media into the tube and invert to mix but do not allow the media to remain at room temperature for long since it will begin to harden as it cools.

Now add 75 μl of cells and invert as before.

Note: A slightly different volume of cells may be announced at the beginning of class, depending on exactly how dense the cell stocks are today.

Pour the 15 ml volume into a Petri dish and allow the media to harden on the bench (~30 minutes). Then flip the plate over, tape on the photomask (using lab tape), and move the plates into the 37 °C incubator that is now equipped with a red-filtered lamp.

The dishes will be exposed to red filtered light (0.08-0.15W/m2 650nm range) until tomorrow morning.

The TA will also prepare one plate without a mask that is completely exposed to the light, so you can observe background levels of β-gal production.

For next time

The sequence for the version of Plux-λ in the edge detector construct is given here. For homework, you should carefully annotate where each of the two promoters starts, any -35 and -10 regions (and which promoter they are associated with), and any operators.

For information about the lambda promoter (PR) and operators, refer to the Day 2 introductory material.

For information about the lux promoter (P(luxI)), you can search for primary literature on PubMed, or read about the lux operon on the Registry of Standard Biological parts. Note that the registry may containing some naming errors.

Briefly, explain the function of what is called the lux box (and be sure to include it in your annotation).

Based on your carefully annotated sequence, do you see a design flaw in this hybrid promoter that might explain its behavior?

Your first short reflective piece, on your experience of and participation during Module 1, is due next time. Refer to the Assignments page for a fuller description.